Whole blood/mitogen assay for the early detection of a subject infected with hepatitis c virus and kit

ABSTRACT

This invention provides a method of detection of antibodies directed against Hepatitis C virus, comprising the following steps: a) obtaining a whole blood sample from the subject; b) incubating the whole blood sample in a culture in the presence of media containing mitogen, so as to induce polyclonal activation of lymphocytic cells to produce antibodies to Hepatitis C virus; c) exposing the resultant culture of step b) to a Hepatitis C virus antigen, thereby allowing an antigen-antibody immune complex to form; and d) detecting the presence of antigen-antibody immune complex of step c); wherein the presence of Hepatitis C virus specific antibodies is indicative of the subject being exposed to Hepatitis C virus.  
     Lastly, this invention provides a kit for the detection of specific Hepatitis C virus specific antibodies from the subject.

[0001] This application is a continuation-in-part application of U.S. Ser. No. 08/275,933, filed Jul. 15, 1994, which is a continuation of U.S. Ser. No. 08/275,933, filed Jul. 15, 1994, a continuation of U.S. Ser. No. 08/095,824, filed Aug. 21, 1993, a continuation of U.S. Ser. No. 07/797,730, filed Nov. 25, 1991, which are hereby incorporated herewith.

[0002] Throughout this application, various publications may be referenced by Arabic numerals in brackets. Full citations for these publications may be found at the end of the specification preceding the claims. The disclosures of the publications cited herein are in their entirety hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.

BACKGROUND OF THE INVENTION

[0003] Viral hepatitis resulting from a virus other than hepatitis A virus (HAV) and hepatitis B virus (HBV) has been referred to as non-A, non-B hepatitis (NANBH). Also, NANBH or ET-NANBH, is contracted predominantly in poor-sanitation areas where food and drinking water have been contaminated by fecal matter. The molecular cloning of a portion of this virus, referred to as the hepatitis E virus (HEV), has been described (Reyes et al.).

[0004] The genome of HCV, the main cause of transfusion-related non-A, non-B hepatitis, has been cloned (Choo, W. L. et al., Science, 244, 359-362, 1989).

[0005] Synthesis of cDNA by reverse transcription of viral RNA and amplification by PCR of the nucleotide sequence of the HCV cDNA has been useful for detecting HCV genome (Weiner, A. J. et al., Lancet, 335, 1-3, 1990). Immunodiagnostic assays for antibodies against HCV proteins have also been developed (Kuo G. et al., Science, 244, 362-364, 1989). Studies with chimpanzees have provided evidence that the virus is probably enveloped (Feinstone, S. M. et al., Infect.

[0006] Immun, 41, 816-821, 1983), and is approximately 30-60 nm in diameter (He, L. F. et al., Infect Dis., 156, 636-640, 1987).

[0007] Hillings et al. reported on the transmission of non-A, non-B hepatitis (presumably hepatitis C) to chimpanzees by inoculation with leucocytes, most probably T leucocytes, derived from either acutely or chronically infected patents or chimpanzees (Hellings, J. A. et al., J. Virol. Methods, 1985).

SUMMARY OF THE INVENTION

[0008] This invention provides a method of detecting a subject infected with a Hepatitis C virus, comprising the following steps: a) obtaining a whole blood sample from the subject; b) incubating the whole blood sample in a culture in the presence of a media containing a mitogen, so as to induce polyclonal activation of lymphocytic cells leading to antibody production; c) exposing the resultant culture of step b) to an specific Hepatitis C virus antigen, thereby allowing an antigen-antibody immune complex to form; d) detecting the antigen-antibody immune complex of step c); wherein the presence of Hepatitis C virus specific antibody is indicative of the subject being infected with a Hepatitis C virus

[0009] Lastly, this invention provides a kit for the detection of specific Hepatitis C virus antibodies from the subject.

DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention relates to an improved assay for detecting a subject infected with Hepatitis C virus. This invention is contemplated for use as: 1) a diagnostic to determine if an individual has been infected with Hepatitis C virus, 2) a prognostic indicator, and 3) a method to monitor the effectiveness of anti-viral treatment (or effectiveness of a new potential anti-Hepatitis C virus drug.

[0011] This invention provides an in vitro method for the detection of antibodies directed against a Hepatitis C virus in a sample obtained from a subject, comprising the following steps: a) obtaining a whole blood sample from the subject; b) incubating the whole blood sample in a culture in the presence of a mitogen containing media,so as to induce polyclonal activation of lymphocytic cells leading to antibody production; c) exposing the resultant culture of step b) to an Hepatitis C virus antigen, thereby allowing an antigen-antibody immune complex to form; d) detecting the antigen-antibody immune complex of step c); wherein the presence of Hepatitis C virus specific antibodies is indicative of the subject being exposed to Hepatitis C virus.

[0012] In one embodiment the specific antibodies producing cells are detected. The culture of step b) may result in a supernatant, and the supernatant may exposed to an Hepatitis C virus antigen, thereby allowing an antigen-antibody immune complex to form. Also, leukocytes or cellular fraction of the culture may be used.

[0013] This invention provides a method of detecting a subject infected with a Hepatitis C virus, comprising the following steps: a) obtaining a whole blood sample from the subject; b) incubating the whole blood sample in a culture in the presence of a media containing a mitogen, so as to induce polyclonal activation of lymphocytic cells leading to antibody production; c) treating the cells so as to recover nucleic acid sequences; d) contacting the resulting nucleic acid sequences with single-stranded labeled oligonucleotide primers, the primers being capable of specifically hybridizing with the nucleic acid sequence of the antibody produced as a result of exposure to Hepatitis C virus, under hybridizing conditions; and e) detecting the presence of the amplification product, the presence thereof being indicative of the presence of the Hepatitis C virus.

[0014] The cells of step c) may be treated so as to expose nucleic acid sequences of the cell. Methods of exposure are known to those skilled in the art. Alternatively the resulting nucleic acid sequences of step d) may be amplified by a pair of primers which hybridizes to the nucleic acid sequence so as to obtain a double-stranded amplification product. Amplification product may be detected.

[0015] In another embodiment primers contact the resulting nucleic acid sequences capable of hybridizing with nucleic acid sequence at the edge of the variable region, under hybridizing conditions and specifically detected.

[0016] This invention provides a method of detecting a subject infected with a Hepatitis C virus, comprising the following steps: a) obtaining a whole blood sample from the subject; b) incubating the whole blood sample in a culture in the presence of a media containing a mitogen, so as to induce polyclonal activation of lymphocytic cells leading to antibody production; c) treating the cells so as to separately recover nucleic acid sequences; d) contacting the resulting nucleic acid sequences with multiple pairs of single-stranded labeled oligonucleotide primers, each such pair being capable of specifically hybridizing with the nucleic acid sequence of the antibody produced as a result of exposure to Hepatitis C virus, under hybridizing conditions; e ) amplifying any nucleic acid sequences to which a pair of primers hybridizes so as to obtain a double-stranded amplification product; f) treating any such double-stranded amplification product so as to obtain single-stranded nucleic acid sequences therefrom; g) contacting any resulting single-stranded nucleic acid sequences with a labeled oligonucleotide probes, being capable of specifically hybridizing with such the Hepatitis C virus specific antibody, under hybridizing conditions; h) contacting any resulting hybrids with a detectably marked tag which is capable of specifically forming a complex with the labeled-probe, when the probe is present in such a complex, under complexing conditions; and i) detecting the presence of any resulting complexes, the presence thereof being indicative of the presence of the Hepatitis C virus.

[0017] As defined herein “sample” refers to any sample obtained from an organism. Examples of biological samples include body fluids and tissue specimens. The source of the sample may be derived from such physiological media as blood, serum, plasma, breast milk, pus, tissue scrapings, washings, urine, tissue, such as lymph nodes, or the like.

[0018] The subjects may be a mammal, or more specifically a human, horse, pig, rabbit, dog, monkey, goat, sheep, cat, cow, or rodent. In the preferred embodiment the subject is a human.

[0019] “Mitogens” as defined herein means any material that activates lymphocytic cells, so as to secrete or produce specific antibodies. In one embodiment the mitogen is a B cell activator, such as pokeweed mitogen, a lectin, a bacterial endotoxin, an antigen, lipid A, or a lymphokine. In another embodiment the mitogen is a superantigen such as a toxin from bacteria which include staphylococci and staphylococci A toxins (30 KD toxins), enterotoxins A, B, C1, C2, D, E (from Staphylococcus aureus), exotoxins A,B,C, and exfoliative toxin A, B. In another embodiment, the mitogen is a gram-negative LPS sequence.

[0020] In another embodiment the mitogen is a peptidoglycan from both gram negative and gram positive bacteria, for example, toxic shock syndrome toxin TSST-1, ExFT, MAM, Strep M., or a Gram-negative lipopolysacchride (LPS) sequences. In another embodiment the mitogen is herpesviruses such as Epstein-Barr Virus(EBV), a retrovirus, mouse mammary tumor virus (MMTV), picomavirus(rats) Coxsackie virus, mumps and measles viruses and Mtv virus (1 -9, 11, 13, 43). In another embodiment the mitogen is heat shock proteins(HSP). In another embodiment the mitogen is an antibody which includes but is not limited to: Anti CD3 antibodies, Anti TCR (T cell Receptor), Anti IgM, Anti IgD, Anti CD28 in both soluble form or bound. It is contemplated that interleukines, such as IL-4, either alone or in conjunction with additional factors may be added. In another embodiment the mitogen is phorbol ester such as phorbol myristate acetate, PMA with calcium ionophore and IL-4. In another embodiment pharmacological activators (such as diacylglycerol) that work through paths such as the PIP2 derived second messenger path. In another embodiment the mitogen is a lectin including Pokeweed mitogen (PWM) and similar acting mitogens. In the preferred embodiment Pokeweed mitogen is used.

[0021] As used herein, “whole blood” means blood collected from an animal or human. The whole blood contains red blood cells which may be lysed while maintaining the viability of the remaining white blood cells. Whole blood may be collected with heparin, EDTA, citrate or any other substance that prevents coagulation and clotting.

[0022] In one embodiment, the optimal concentration of mitogen is easily determined without undue experimentation by one of ordinary skill in the art. With regard to the preferred mitogen, pokeweed mitogen, the preferred concentration range is between approximately 1:100 and 1:1600 dilutions of stock PWM. The most preferred concentration range is between approximately 1:200 and 1:1:400 dilutions of stock PWM. The preferred source of the stock PWM is GIBCO, New York, N.Y. The lyophilized PWM is reconstituted with 5 ml of distilled water to make the stock solution.

[0023] The concentration of pokeweed mitogen may range from about 0.05-50.0 ul/ml. The concentration range may be from about 0.1-0.5 ul/ml. The preferred concentration is 0.2 ul/ml. If the mitogen is Wheat germ agglutination the concentration range is about 0.1-2.5 ul/ml. If the mitogen is Sac Cowan I mitogen the concentration range is 1:200-1:2000 dilution.

[0024] As defined herein, “culture medium” means any medium than can be used to sustain a sample to practice the present invention, including but not limited to RPMI 1640 (GIBCO, New York, N.Y.) with or without fetal calf serum, preferably supplemented with appropriate antibiotics and glutamine. Other culture media which may be used in practicing the present invention include, but are not limited to, Eagles, Dulbecco's, McCoy's, Media 199, Waymouth's media, and serum free medium with or without supplement. In another embodiment the mitogen is without media.

[0025] The nucleic acid sequence referred herein may be DNA, RNA or cDNA.

[0026] Amplification is carried out using the polymerase chain reaction and a single or plurality of primer sets so as to provide PCR products of different lengths. The plurality of primer sets are amplified together by PCR. Each primer set is amplified separately by PCR.

[0027] If one employs two different primer sets, the present invention contemplates that the first primer set is capable of generating a product of a length short enough to be essentially transparent to the addition of the addition compounds to the nucleic acid under a defined set of amplification conditions. That is regardless of the efficiency of the covalent addition of addition compounds, the length of the product is sufficient such that amplification product will be detected.

[0028] The second primer set is also capable of generating a product of a length long enough to be affected-not completely inhibited but inhibited in part-by the addition of the addition compounds to the nucleic acid of the nucleic acid-containing antibodies with the same amplification conditions as above. This being so the efficiency of the covalent addition of addition compounds will be reflected in the amount of amplification product detected.

[0029] Where three different primer sets are used, the present invention contemplates that the first primer set is capable of generating a product of a length short enough to be essentially transparent to the addition of the addition compounds to the nucleic acid under a defined set of amplification conditions. In other words regardless of the efficiency of the covalent addition of addition compounds, the length of the product is sufficient such that amplification product will be detected. The second primer set is also capable of generating a product of a length long enough to be affected-not completely inhibited but inhibited in part-by the addition of the addition compounds to the nucleic acid of the nucleic acid-containing antibodies with the same amplification conditions as above. Again the efficiency of the covalent addition of addition compounds will be reflected in the amount of amplification product detected. The third primer set is capable of generating a product of a length long enough to be completely inhibited by the addition of addition compounds to the nucleic acid of the nucleic-acid containing antibodies. Covalent addition of the addition compounds will be reflected by the complete absence of measurable amplification product.

[0030] For example, after amplification, a portion of the PCR reaction mixture can be separated and subjected to hybridization with an end-labeled nucleotide probe, such as a 32P labeled adenosine triphosphate end-labeled probe. In PCR, an end-labeled oligonucleotide probe hybridizes in solution to a region of the amplified sequence and, in the process, reconstitutes a specific endonuclease site. Thus, hybridization of the labeled probe with the amplified Hepatitis C virus antibodies sequence yields a double-stranded DNA form that is sensitive to selective restriction enzyme digestion. After restriction with an endonuclease, the resulting samples can be analyzed on a polyacrylamide gel, and autoradiograms of the portion of the gel with the diagnostic labeled fragment can be obtained. The appearance of a diagnostic fragment (e.g. 10-15 bases in length) in the autoradiogram indicates the presence of Hepatitis C virus sequences in the PBMCs.

[0031] This invention provides a method for the detection of Hepatitis C virus, comprising: a) obtaining a whole blood sample from the subject; b) incubating the whole blood sample in a culture in the presence of a media containing a mitogen, so as to induce polyclonal activation of lymphocytic cells leading to antibody production; c) treating the cells so as to separately recover nucleic acid sequences; d) performing reverse transcription of an RNA of a Hepatitis C virus in a sample to produce a DNA copy; e) performing polymerase chain reaction amplification of the DNA copy of the RNA genome to produce a plurality of DNA copies; f) performing hybridization of the DNA copies to a plurality of complementary DNA probes to detect the DNA content of the sample; thereby detecting Hepatitis C virus.

[0032] This invention provides a method for amplifying a target RNA sequence in a sample, comprising: a) obtaining a whole blood sample from the subject; b) incubating the whole blood sample in a culture in the presence of a media containing a mitogen, so as to induce polyclonal activation of lymphocytic cells leading to antibody production; c) exposing the cells so as to separately recover nucleic acid sequences of the Hepatitis C virus antibody; d) treating the sample in a reaction mixture comprising a first and second primer, wherein the first primer is sufficiently complementary to the target RNA to hybridize therewith and initiate synthesis of a cDNA sequence complementary to the target RNA, and the second primer is sufficiently homologous to the target RNA to hybridize to the cDNA and initiate synthesis of an extension product, and a thermostable DNA polymerase in the presence of all four deoxyribonucleoside triphosphates, in an appropriate buffer, wherein the buffer comprises Mn+2, at a temperature sufficient for the thermostable DNA polymerase to initiate synthesis of an extension product of the first primer to provide a cDNA sequence complementary to the target RNA; e) treating the reaction mixture at an appropriate temperature to provide single-stranded cDNA; f) treating the reaction mixture at an appropriate temperature for the thermostable DNA polymerase to initiate synthesis of an extension product of the second primer to provide a double-stranded cDNA sequence; and g) amplifying the double-stranded cDNA sequence of step e) by a polymerase chain reaction. In a preferred embodiment, the buffer comprises manganese acetate (also written Mn(OAc)2 or Mn(CH3CO2)2), Bicine-KOH (Bicine is N,N-Bis(2-Hydroxyethyl)glycine), and potassium acetate (also written KOAc or KCH3CO2).

[0033] Nucleotide sequences of specific antibodies produced as a result of Hepatitis C virus infection and/or exposure can be employed in a DNA amplification process known as the “polymerase chain reaction (PCR), which is useful for amplifying specific regions of Hepatitis C virus.

[0034] “PCR” refers to a process of amplifying one or more specific nucleic acid sequences, wherein (1) oligonucleotide primers which determine the ends of the sequences to be amplified are annealed to single-stranded nucleic acid in a test sample, (2) a nucleic acid polymerase extends the 3′ ends of the annealed primers to create a nucleic acid strand complementary in sequence to the nucleic acid to which the primers were annealed, (3) the resulting double-stranded nucleic acid is denatured to yield two single-stranded nucleic acids, and (4) the processes of primer annealing, primer extension, and product denaturation are repeated enough times to generate easily identified and measured amounts of the sequences defined by the primers. Practical control of the sequential annealing, extension, and denaturation steps is exerted by varying the temperature of the reaction container, normally in a repeating cyclical manner. Annealing and extension occur optimally in the 40° C. to 80° C. temperature range (exact value depending on primer concentrations and sequences), whereas denaturation requires temperatures in the 80° C. to 100° C. range (exact value depending on target sequence and concentration).

[0035] DNA amplification procedures by PCR are well known and are described in U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, each of which is incorporated herein by reference. For ease of understanding the advantages provided by the present invention, a summary of PCR is provided. PCR requires two primers that hybridize with the double-stranded target nucleic acid sequence to be amplified. In PCR, this double-stranded target sequence is denatured and one primer is annealed to each strand of the denatured target. The primers anneal to the target nucleic acid at sites removed from one another and in orientations such that the extension product of one primer, when separated from its complement, can hybridize to the other primer. Once a given primer hybridizes to the target sequence, the primer is extended by the action of a DNA polymerase. The extension product is then denatured from the target sequence, and the process is repeated. One particular method for minimizing the effects of cross contamination of nucleic acid amplification is described in U.S. Pat. No. 5,035 is incorporated herein by reference.

[0036] The PCR technique useful for determining whether seropositive or seronegative persons have detectable levels of specific antibodies. In PCR techniques, oligonucleotide primers to the specific antibody produced from Hepatitis C virus exposure complementary to the two 3′ borders of the DNA region to be amplified are synthesized. The polymerase chain reaction is then carried out using the two primers. See PCR Protocols: A Guide to Methods and Applications [PCR Protocols: A Guide to Methods and Applications. (1990) Innis, M., Gelfand D., Sninsky, J. and White, T., eds., Academic Press, San Diego]. Following PCR amplification, the PCR-amplified regions of a viral DNA can be tested for their ability to hybridize to the three specific nucleic acid probes listed above. Alternatively, hybridization of a viral DNA to the above nucleic acid probes can be performed by a Southern blot procedure without viral DNA amplification and under stringent hybridization conditions as described herein. U.S. Pat. No. 5,494,810 Barany, Francis,et al. “Polymerase chain reaction (PCR)” refers to a patented process (described in U.S. Pat. Nos. 4,683,202 and 4,683,195) for the exponential amplification of a specific DNA fragment by utilizing two oligonucleotide primers that hybridize to opposite strands and flank the region of interest in a target DNA are incorporated by reference. Also, those assays disclosed in U.S. patents: U.S. Pat. No. 4,459,359 is incorporated by reference.

[0037] Choosing PCR primer sequences, preparing PCR reagents and reaction mixtures, and designing and running PCR reactions are well known procedures in the PCR art. In the event that nucleic acid amplification is performed on suspended cells in a standard PCR tube, the cells are treated like any conventional PCR test sample: diluted into reaction mixture shortly before amplification is started, at a total cell number ranging from approximately 100 to approximately 10⁶.

[0038] If multiple samples are amplified simultaneously in different tubes, a fresh sampler tip is used to add the missing reagent(s) to each tube, to prevent cross-contamination. After all tubes have been prepared and capped, the standard three-temperature thermal cycle program of denaturation, annealing, and extension for approximately 10 to 40 cycles is performed under thermal cycler microprocessor control. Alternatively, and often preferably, a series of two-temperature cycles can be run wherein annealing and extension are performed at a single temperature, normally optimized for stringent annealing of primer to template. Because reaction rates may be somewhat retarded with cellular preparations as compared to cell-free nucleic acids, it may be necessary to increase the durations of the denaturation, anneal, extend, or anneal-extend cycle segments as much as several-fold from values standard when the test sample contains cell-free nucleic acid. This adjustment is easily performed by looking for conditions which maximize the intensity of the signal seen during amplified nucleic acid detection or which minimize the number of cycles needed to reach a given signal intensity. A similar optimization procedure can be used for MgCl2, dNTP, primer, and enzyme concentrations in the reaction mixture; these parameters often show different optima for different targets, and also may be affected when amplification occurs within fixed cells.

[0039] Primer pairs of known sequence positioned 10-300 base pairs apart that are complementary to the plus and minus strands of the DNA to be amplified can be prepared by well known techniques for the synthesis of oligonucleotides. One end of each primer can be extended and modified to create restriction endonuclease sites when the primer is annealed to the nucleic acid sequence of specific antibodies. The PCR reaction mixture can contain the DNA of the specific antibodies, the DNA primer pairs, four deoxyribonucleoside triphosphates, MgCl2, DNA polymerase, and conventional buffers The DNA can be amplified for a number of cycles. It is generally possible to increase the sensitivity of detection by using a multiplicity of cycles, each cycle consisting of a short period of denaturation of the DNA of the specific antibodies at an elevated temperature, cooling of the reaction mixture, and polymerization with the DNA polymerase. Oligonucleotide primers and probes are known to those skilled in the art. For example primers which hybridize to specific antibodies produced as a result of infection or exposure to Hepatitis C virus include:

[0040] Oligonucleotides for use as probes or PCR primers are chemically synthesized according to the solid phase phosphoramidite triester method first described by Beaucage and Carruthers [Beaucage and Carruthers (1981) Tetrahedron Lett. 22:1859-1862.] using an automated synthesizer, as described in Needham-VanDevanter [Needham-VanDevanter, D. R., et al., (1984) Nucleic Acids Res. 12:6159-6168]. Purification of oligonucleotides is by either native acrylamide gel electrophoresis or by anion-exchange HPLC as described in Pearson, J. D. and Regnier, F. E. [Pearson, J. D., and Regnier, F. E., (1983) J. Chrom. 255:137-14976.]. The sequence of the synthetic oligonucleotide can be verified using the chemical degradation method of Maxam, A. M. and Gilbert, W. [Maxam, A. M. and Gilbert, W. Methods in Enzymology (1980) Grossman, L. and Moldave, D., eds., Academic Press, New York, 65:499-560.]. Tavernarakis, N., U.S. Pat. No. 5,569,582 is directed to amplification and detection of Hepatitis C virus nucleic acid is hereby incorporated by reference.

[0041] The present invention has applicability to diagnosing diseases by the amplification and subsequent detection of nucleic acid sequences of anti viral specific antibodies. “Amplification” is a special case of nucleic acid replication involving template specificity. It is to be contrasted with non-specific template replication (i.e., replication that is template-dependent but not dependent on a specific template). Template specificity is here distinguished from fidelity of replication (i.e., synthesis of the proper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-) specificity. Template specificity is frequently described in terms of “target” specificity. Target sequences are “targets” in the sense that they are sought to be amplified or detected preferentially in the presence of other non-target nucleic acid sequences. Amplification techniques have been designed primarily for the detection of specific target sequences. Template specificity is achieved, in most amplification techniques, by the choice of enzyme. Amplification enzymes are enzymes that, under the conditions in which they are used, will process only specific sequences of nucleic acid in a heterogenous mixture of nucleic acid.

[0042] In the case of T4 DNA ligase, the enzyme will not ligate the two oligonucleotides where there is a mismatch between the oligonucleotide substrate and the template at the ligation junction. D. Y. Wu and R. B. Wallace, Genomics 4:560 (1989). Finally, Taq polymerase, by virtue of its ability to function at high temperature, is found to display high specificity for the sequences bounded and thus defined by the primers; the high temperature results in thermodynamic conditions that favor primer hybridization with the specific target sequences and not hybridization with non-target sequences. R. K. Saiki in PCR Technology, Principles and Applications for DNA Amplification (H. A. Erlich, Ed.), pp. 7-16 (1989).

[0043] Some amplification techniques take the approach of amplifying and then detecting target; others detect target and then amplify probe. Regardless of the approach, the sample containing nucleic acid must be free of inhibitors for amplification to occur at high efficiency.

[0044] Amplification “reagents” are defined as those reagents (primers, deoxyribonucleotide triphosphates, etc.) needed for amplification except for nucleic acid and the amplification enzyme. Typically, amplification reagents along with other reaction components are placed and contained in a reaction vessel (test tube, microwell, etc.).

[0045] The preferred lysing agent is protease K. Protease K is a proteolytic enzyme from Tritirachium album. It is particularly useful in the present invention because it has no significant DNase activity and, therefore, does not degrade nucleic acid which would prevent amplification. It is also attractive because it is inexpensive and commercially available (e.g., Sigma, St. Louis, Mo., U.S.A., catalogue No. p4914 “Proteinase K”). Various treatment conditions using protease K have been found useful. It is preferred that a high concentration of protease K (e.g., 1.5-2.5 mg/ml) be used for short (5-10 minutes) incubation periods to completely degrade cellular and viral protein and expose viral nucleic acid for amplification. When lower concentrations of protease K (e.g., 0.5 mg/ml) are used, longer incubation periods (30-60 minutes) are required to achieve the same effect. Other lysis approaches are also contemplated, including lysis by heating.

[0046] The present invention also contemplates labeling methods wherein the oligonucleotide probe sequences have at least one label attached or integrated into its structure. Labels are generally intended to facilitate the detection of the virus. Labels are chosen from the group consisting of enzymes, fluorophores, high-affinity conjugates, chemiphores and radioactive atoms (“radiolabels”). While other labels may be used, the present invention contemplates: 1) the enzymes alkaline phosphatase, beta -galactosidase and glucose oxidase; 2) the affinity conjugate system of biotin-avidin; 3) the fluorophore that is fluorescein; 4) the chemiphore that is luminol; and 5) the preferred radiolabels 3H, 14C and 32P.

[0047] “Detection” of PCR-amplified nucleic acid refers to the process of observing, locating, or quantitating an analytical signal which is inferred to be specifically associated with the product of PCR amplification, as distinguished from PCR reactants. The analytical signal can result from visible or ultraviolet absorbance or fluorescence, chemiluminescence, or the photographic or autoradiographic image of absorbance, fluorescence, chemiluminescence, or ionizing radiation. Detection of in situ PCR products involves microscopic observation or recording of such signals. The signal derives directly or indirectly from a molecular “tag” attached to a PCR primer or dNTP or to a nucleic acid probe, which tag may be a radioactive atom, a chromophore, a fluorophore, a chemiluminescent reagent, an enzyme capable of generating a colored, fluorescent, or chemilurninescent product, or a binding moiety capable of reaction with another sequence or particle which directly carries or catalytically generates the analytical signal. Common binding moieties are biotin, which binds tightly to streptavidin or avidin, digoxigenin, which binds tightly to anti-digoxigenin antibodies, and fluorescein, which binds tightly to anti-fluorescein antibodies. The avidin, streptavidin, and antibodies are easily attached to chromophores, fluorophores, radioactive atoms, and enzymes capable of generating colored, fluorescent, or chemilurninescent signals.

[0048] RNA is prepared by any number of methods; the choice may depend on the source of the sample and availability. Methods for preparing RNA are described in Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier, N.Y., Chapter 11; Ausubel et al., 1987, Current Protocols in Molecular Biology, Chapter 4, John Wiley and Sons, NY; Kawasaki and Wang, 1989, PCR Technology, ed. Erlich, Stockton Press NY; Kawasaki, 1990, PCR Protocols: A Guide to Methods and Applications, Innis et al. eds. Academic Press, San Diego; and Wang and Mark, 1990, PCR Protocols: A Guide to Methods and Applications, Innis et al. eds. Academic Press, San Diego; all of which are incorporated herein by reference. As noted above, the probe will be capable of specific hybridization to a specific antibody produced as a result of infection or exposure of Hepatitis C virus nucleic acid. Such “specific hybridization” occurs when a probe hybridizes to a target nucleic acid, as evidenced by a detectable signal, under conditions in which the probe does not hybridize to other nucleic acids (e.g., animal cell or other bacterial nucleic acids) present in the sample. A variety of factors including the length and base composition of the probe, the extent of base mismatching between the probe and the target nucleic acid, the presence of salt and organic solvents, probe concentration, and the temperature affect hybridization, and optimal hybridization conditions must often be determined empirically. For discussions of nucleic acid probe design and annealing conditions, see, for example, Ausubel, F., et al., Methods in Enzymology [Methods in Enzvmology Vol. 152, (1987) Berger, S. and Kimmel, A. ed., Academic Press, New York] or Hybridization with Nucleic Acid Probes all of which are incorporated herein by reference.

[0049] High stringent hybridization conditions are selected at about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.

[0050] Typically, stringent conditions will be those in which the salt concentration is at least about 0.02 molar at pH 7 and the temperature is at least about 60° C. As other factors may significantly affect the stringency of hybridization, including, among others, base composition and size of the complementary strands, the presence of organic solvents, ie. salt or formamide concentration, and the extent of base mismatching, the combination of parameters is more important than the absolute measure of any one. For Example high stringency may be attained for example by overnight hybridization at about 68° C. in a 6× SSC solution, washing at room temperature with 6× SSC solution, followed by washing at about 68° C. in a 6× SSC in a 0.6× SSX solution.

[0051] Hybridization with moderate stringency may be attained for example by: 1) filter pre-hybridizing and hybridizing with a solution of 3× sodium chloride, sodium citrate (SSC), 50% formamide, 0.1M Tris buffer at Ph 7.5, 5× Denhardt's solution; 2.) pre-hybridization at 37° C. for 4 hours; 3) hybridization at 37° C. with amount of labeled probe equal to 3,000,000 cpm total for 16 hours; 4) wash in 2× SSC and 0.1% SDS solution; 5) wash 4× for 1 minute each at room temperature at 4× at 60° C. for 30 minutes each; and 6) dry and expose to film.

[0052] The phrase “selectively hybridizing to” refers to a nucleic acid probe that hybridizes, duplexes or binds only to a particular target DNA or RNA sequence when the target sequences are present in a preparation of total cellular DNA or RNA. By selectively hybridizing it is meant that a probe binds to a given target in a manner that is detectable in a different manner from non-target sequence under high stringency conditions of hybridization. in a different “Complementary” or “target” nucleic acid sequences refer to those nucleic acid sequences which selectively hybridize to a nucleic acid probe. Proper annealing conditions depend, for example, upon a probe's length, base composition, and the number of mismatches and their position on the probe, and must often be determined empirically. For discussions of nucleic acid probe design and annealing conditions, see, for example, Sambrook et al., [Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed.), Cold Spring Harbor Laboratory, Vols. 1-3.] or Ausubel, F., et al., [Ausubel, F., et al. (1987) Current Protocols in Molecular Biology, New York.].

[0053] DNA virus nucleic acid rearrangements/mutations may be detected by Southern blotting, single stranded conformational polymorphism gel electrophoresis (SSCP), PCR or other DNA based techniques, or for RNA species by Northern blotting, PCR or other RNA-based techniques.

[0054] As defined herein nucleic acid probes may be DNA or RNA fragments. DNA fragments can be prepared, for example, by digesting plasmid DNA, or by use of PCR, or synthesized by either the phosphoramidite method described by Beaucage and Carruthers, or by the triester method according to Matteucci, et al., [Matteucci, et al. (1981) Am. Chem. Soc. 103:3185.], both incorporated herein by reference. A double stranded fragment may then be obtained, if desired, by annealing the chemically synthesized single strands together under appropriate conditions or by synthesizing the complementary strand using DNA polymerase with an appropriate primer sequence. Where a specific sequence for a nucleic acid probe is given, it is understood that the complementary strand is also identified and included. The complementary strand will work equally well in situations where the target is a double-stranded nucleic acid. It is also understood that when a specific sequence is identified for use a nucleic probe, a subsequence of the listed sequence which is 25 base pairs or more in length is also encompassed for use as a probe.

[0055] Examples of such antigens and specific antibodies which are immunoreactive with sera from patients infected with parenterally transmitted non-A, non-B hepatitis (PT-NANBH) virus, to polynucleotide sequences which encode the peptides, to an expression system capable of producing the peptides, and to methods of using the peptides for detecting PT-NANBH infection in human sera are known to those skilled in the art. Specifically, this invention contemplates use of the HCV genome core, NS4 regions, surface-bound HCV antigen, 409-1-1 antigens (409-1-1(abc), 409-1-1(c-a), and related antigens. Yoshikura, Hiroshi, et al, U.S. Pat. No. 5,552,310 Replication of Hepatitis C virus genome and identification of virus having high infectivity; Reyes, Gregory,et al., U.S. Pat. Nos. 5,538,865, 5,443,965 and 5,436,318 Hepatitis C virus epitopes; Resnick, Robert M., et al., U.S. Pat. No. 5,527,669, Methods, primers and probes for detection of hepatitis C and novel variants; and Wang, Chang Y., U.S. Pat. No. 5,436,126 Synthetic peptides specific for the detection of specific antibodies to HCV, diagnosis of HCV infection.

[0056] Further, antigens are known to those skilled in the art. For example, the following antigens may be used SOD/HCV polypeptide c100-3 (363 AA); Polypeptide 2nd; and capsoid peptides.

[0057] This invention provides a kit for the detection of specific Hepatitis C virus antigen from a subject, comprising: a container for collecting whole blood samples, wherein the container contains a media containing mitogen, effective to induce polyclonal activation of lymphocytic cells leading to antibody production and an assay for the detection of the specific Hepatitis C virus antibody.

[0058] This invention provides a kit for the detection of specific human immunodeficiency virus antigen from a subject, comprising: a vacuum sealed container for collecting whole blood samples, wherein the container contains a media containing mitogen, effective to induce polyclonal activation of lymphocytic cells leading to antibody production. Single or multiple antigens may be bound to the container.

[0059] In accordance with the present invention, a blood sample is drawn into a test tube, such as a vacutube, containing an effective concentration of a solution of a mitogen, such as pokeweed mitogen. The blood sample to be tested is cultured in vitro in the presence of the pokeweed mitogen. Other activators of B lymphocytic cells or the humoral system which leads to antibody production or secretion and activators of the Th₂ type immune response may be used in place of or in addition to the pokeweed mitogen to achieve the same function. After incubation, an aliquot is taken from the top of the fluid and is then assayed for the presence of desired antigen using standard ELISA procedures and/or Western Blot analysis. If the sample is to be assayed at a later date, the blood may be centrifuged and the supernatant fluid may be collected, frozen and stored.

[0060] Results may be verified utilizing the technique of polymerase chain reaction (PCR)/FACS. Cellular fractions may be used with PCR and Elispot.

[0061] The method of the present invention includes optionally separating the blood cells from the fluid portion of the blood so that the presence of antibody can be determined. The separation of the blood cells from the fluid portion of the blood can be done by any of several methods well known to those of ordinary skill in the art, including centrifugation, filtration or density gradient. It is to be understood, that the blood cells do not need to be physically separated from the fluid. After incubation of the whole blood with the mitogen, fluid from the top of the blood can easily be extracted and tested for antibody. Optionally, the red blood cells can be lysed either by mild osmotic shock or with a mild detergent. In this way, the white blood cells remain viable.

[0062] In one embodiment of the present invention, whole blood is collected in a blood collection tube containing culture medium and mitogen. The blood samples are then incubated with an approximately 1:500 final dilution of pokeweed mitogen at a concentration of 2×10⁶viable lymphocytes per ml for 1-12 days at 37° C. in a 7% CO₂ humidified atmosphere. The blood may be centrifuged and the supernatant fluid is collected and assayed immediately within approximately 12 hours or frozen for later testing days for reactive specific antibodies by ELISA/Elispot and/or Western blot techniques. In the alternative, an aliquot of fluid or cells or cellular components may be taken directly from the sample.

[0063] The present invention also includes a kit comprising a blood collection container containing an effective concentration of mitogen therein. The container can optionally contain a culture medium. The preferred container is a test tube. The blood collection container can be plastic, glass, or any other material that is compatible with culturing blood. It is to be understood that the present invention also includes blood containing means other then a blood collection tube including, but not limited to, microtiter plates containing wells in which the blood can be incubated, tissue culture flasks, glass flasks such as an Erlenmeyer flask, and any other container in which the blood can be cultured.

[0064] Antigens which specifically bind specific antibodies of the to Hepatitis C virus are known to those skilled in the art. Further, antibodies may be used as tags to bind to the antigen. In one embodiment, the antibody is a monoclonal antibody. In another embodiment the antibody is a polyclonal antibody.Antibodies include but are not limited to: IgG and subsets, IgA, IgE, IgM, IgD.

[0065] The antibody or DNA sequence may be labeled with a detectable marker including, but not limited to: a radioactive label, or a colorimetric, a luminescent, or a fluorescent marker, or gold. Radioactive labels include, but are not limited to ³H, ¹⁴C, ³²P, ³³,³⁵, S³⁶, Cl⁵¹, Cr⁵⁷, Co⁵⁹, Fe⁹⁰, Y¹²⁵, I¹³¹, I¹⁸⁶, and Re. Fluorescent markers include but are not limited to: fluorescein, rhodamine and auramine. Colorimetric markers include, but are not limited to: biotin, and digoxigenin. Methods of producing the polyclonal or monoclonal antibody are known to those of ordinary skill in the art.

[0066] Further, the antibody or nucleic acid sequence complex may be detected by a second antibody which may be linked to an enzyme, such as alkaline phosphatase or horseradish peroxidase. Other enzymes which may be employed are well known to one of ordinary skill in the art.

[0067] The antigens or antibodies may also be labeled using fluorescent labels, enzyme labels, free radical labels, or bacteriophage labels, using techniques known in the art. Typical fluorescent labels include fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, alophycocyanin, and Texas Red.

[0068] Since specific enzymes may be coupled to other sequences by covalent links, the possibility also exists that they might be used as labels for the production of tracer materials. Suitable enzymes include alkaline phosphatase, beta-galactosidase, glucose-6-phosphate dehydrogenase, maleate dehydrogenase, and peroxidase. Types of enzyme immunoassay are the enzyme-linked immunosorbent assay (ELISA), Elispot, and the homogeneous enzyme immunoassay, also known as enzyme-multiplied immunoassay (EMIT, Syva Corporation, Palo Alto, Calif.). In the ELISA system, separation may be achieved, for example, by the use of antibodies coupled to a solid phase. The EMIT system depends on deactivation of the enzyme in the tracer-antibody complex; the activity can thus be measured without the need for a separation step.

[0069] Additionally, chemiluminescent compounds may be used as labels. Typical chemiluminescent compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters. Similarly, bioluminescent compounds may be utilized for labeling, the bioluminescent compounds including luciferin, luciferase, and aequorin.

[0070] Once labeled, the antibody may be employed to identify and quantify immunologic counterparts (antibody or antigenic polypeptide) utilizing techniques well-known to the art.

[0071] The phrase “specifically binds to an antibody” or “specifically immunoreactive with”, when referring to the antigen, refers to a binding reaction which is determinative of the presence of the Hepatitis C viral antibody. Thus, under designated immunoassay conditions, the specified antigen binds to the Hepatitis C virus specific antibody and does not bind in a significant amount to other antibodies present in the sample. Specific binding to an antibody under such conditions may require an antigen that is selected for its specificity for a particular protein.

[0072] A variety of immunoassay formats may be used to select specific antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane [Harlow and Lane, (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publication, New York.] for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.

[0073] A description of a radioimmunoassay (RIA) may be found in rLaboratory Techniques in Biochemistry and Molecular Biology [Laboratory Techniques in Biochemistry and Molecular Biology (1978) North Holland Publishing Company, New York.], with particular reference to the chapter entitled “An Introduction to Radioimmune Assay and Related Techniques” by Chard, T., incorporated by reference herein.

[0074] A description of general immunometric assays of various types can be found in the following U.S. Pat. No. 4,376,110 (David et al.) or U.S. Pat. No. 4,098,876 (Piasio) and U.S. Pat. No. 4,870,003 (Kortright) are incorporated by reference.

[0075] The presence or concentration of this antigen-antibody complex is determined to detect or quantitate the presence of Hepatitis C virus in the biological sample. Uses of this assay and method include detecting the presence of Hepatitis C virus infection, monitoring the progression of Hepatitis C virus infection, and evaluating the effectiveness of an anti-Hepatitis C virus treatment administered to an animal, such as a human.

[0076] The present invention further provides a kit for detecting the antibody. Particularly, the kit can detect the presence of an antibody specifically reactive with an antigen or an immunoreactive fragment thereof. The kit can include an antigen bound to a substrate, a secondary antigen reactive with the antibody and a reagent for detecting a reaction of the secondary antigen with the antibody. Such a kit can be an antibody capture assay kit, such as an ELISA kit, and can comprise the substrate, primary and secondary antibodies when appropriate, and any other necessary reagents such as detectable moieties, enzyme substrates and color reagents as described above. The antibody capture diagnostic kit can, alternatively, be an immunoblot kit generally comprising the components and reagents described herein. The particular reagents and other components included in the diagnostic kits of the present invention can be selected from those available in the art in accord with the specific diagnostic method practiced in the kit. Such kits can be used to detect the antibody in biological samples, such as tissue and bodily fluid before and after culture obtained from a subject.

[0077] This invention provides a kit for the detection of specific Hepatitis C virus antibody from a subject, comprising: a vacuum sealed container for collecting whole blood samples, wherein the container contains a media containing mitogen, effective to induce polyclonal activation of lymphocytic cells leading to antibody production.

[0078] Detecting the reaction of the antibody (or ligand) with the antigen can be facilitated by the use of an antibody or ligand that is labeled with a detectable moiety by methods known in the art. Such a detectable moiety will allow visual detection of a precipitate or a color change, visual detection by microscopy, or automated detection by spectrometry or radiometric measurement or the like. Examples of detectable moieties include fluorescein and rhodamine (for fluorescence microscopy), horseradish peroxidase (for either light microscopy or electron microscopy and biochemical detection), biotin-strepavidin (for light or electron microscopy) and alkaline phosphatase (for biochemical detection by color change). The detection methods and moieties used can be selected, for example, from the list above or other suitable examples by the standard criteria applied to such selections (Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988).

[0079] In the present invention, detecting the reaction of the ligand with the antigen can be further aided, in appropriate instances, by the use of a secondary antibody or other ligand which is reactive, either specifically with a different epitope or nonspecifically with the ligand or reacted antibody.

[0080] Enzyme immunoassays such as immunofluorescence assays (IFA), photometric assays, enzyme linked immunosorbent assays (ELISA), Elispot, and immunoblotting can be readily adapted to accomplish the detection of the specific antibodies. An ELISA method effective for the detection of the antibody can, for example, be as follows: (1) bind the antigen to a substrate; (2) contact the bound antigen with a biological sample, such as a bodily fluid before and after culture or tissue sample or lymphocytes before and after culture containing the antibody; (3) contact the above with a secondary antibody bound to a detectable moiety (e.g., horseradish peroxidase enzyme or alkaline phosphatase enzyme); (4) contact the above with the substrate for the enzyme; (5) contact the above with a color reagent; (6) observe the color change.

[0081] Where the microbial or viral proteins are labeled, the labels can include radioisotopes, fluorophores, enzymes, luminescers or particles. These and other labels are well known in the art and are described, for example, in the following U.S. Pat. Nos. 3,766,162; 3,791,932; 3,817,837; 3,996,345; and 4,233,402. Assays employing the microbial or viral proteins isolated from the cell lines can be heterogenous, i.e., requiring a separation step, or homogenous. If the assay is heterogenous a variety of separation means can be employed, including centrifugation, filtration, chromatography or magnetism.

[0082] Other detection systems which may also be used include those based on the use of protein A derived from Staphylococcus aureus Cowan strain I, protein G from group C Streptococcus sp. (strain 26RP66), or systems which employ the use of the biotin-avidin binding reaction.

[0083] Another method of immunoenzymatic detection of the presence of specific antibodies directed against one or more of the Hepatitis C virus antigens is the Western blot. The viral antigens are separated electrophoretically and transferred to a nitrocellulose membrane or other suitable support. The body fluid to be tested is then brought into contact with the membrane and the presence of the immune complexes formed is detected by the method already described. In a variation on this methods, purified viral antigen is applied in lines or spots on a membrane and allowed to bind. The membrane is subsequently brought into contact with the body fluid before and after culture to be tested and the immune complexes formed are detected using the previously described techniques.

[0084] The presence of specific antibodies in body fluid before and after culture may also be detected by agglutination. Hepatitis C virus lysates, antigen or purified antigen composition referred to according to this invention, is used to coat, for example, latex particles which form an uniform suspension. When mixed with serum containing specific antibodies to the antigen present, the latex particles are caused to agglutinate and the presence of large aggregates can be detected visually.

[0085] Antigens reactive with specific antibodies of Hepatitis C virus can also be measured by a variety of immunoassay methods. For a review of immunological and immunoassay procedures applicable to the measurement of antibodies by immunoassay techniques, see Basic and Clinical Immunology 7th Edition [Basic and Clinical Immunology 7th Edition D. Stites and A. Terr ed.].

[0086] Hemagglutination Inhibition (HI) and Complement Fixation (CF) which are two laboratory tests that can be used to detect infection with Hepatitis C virus by testing for the presence of antibodies against the virus or antigens of the virus.

[0087] In vitro Diagnostic Assays for the Detection of Hepatitis C Virus

[0088] This invention provides a method of diagnosing Hepatitis C virus in a subject which comprises: a) obtaining a whole blood sample from the subject; b) incubating the whole blood sample in a culture in the presence of a media containing a mitogen, to induce polyclonal activation of lymphocytes; c) obtaining a nucleic acid sequence; d) contacting the nucleic acid sequence with a labeled nucleic acid sequence of at least 15 nucleotides capable of specifically hybridizing with the isolated DNA of the specific antibody, under hybridizing conditions; and e) determining the presence of the nucleic acid sequence hybridized, the presence of which is indicative of Hepatitis C virus in the subject, thereby diagnosing Hepatitis C virus in the subject.

[0089] In one embodiment, the DNA sequence from the Hepatitis C virus is amplified before step (b). In another embodiment PCR is employed to amplify the nucleic acid sequence. Methods of amplifying nucleic acid sequences are known to those skilled in the art.

[0090] A person of ordinary skill in the art would be able to obtain appropriate DNA sample for diagnosing Hepatitis C virus specific antibody in the subject. The DNA sample obtained by the above described method may be cleaved by restriction enzyme. The uses of restriction enzymes to cleave DNA and the conditions to perform such cleavage are well-known in the art.

[0091] In the above described methods, a size fractionation may be employed which is effected by a polyacrylamide gel. In one embodiment, the size fractionation is effected by an agarose gel. Further, transferring the DNA fragments into a solid matrix may be employed before a hybridization step. One example of such solid matrix is nitrocellulose paper.

[0092] A method for detecting the specific antibodies produced as a result of infection or exposure of Hepatitis C virus is the use of PCR and/or dot blot hybridization. The presence or absence of an Hepatitis C virus agent for detection or prognosis, or risk assessment for Hepatitis C virus includes Southern transfers, solution hybridization or non-radioactive detection systems, all of which are well known to those of skill in the art. Hybridization is carried out using probes. Visualization of the hybridized portions allows the qualitative determination of the presence or absence of the causal agent.

[0093] Similarly, a Northern transfer may be used for the detection of message in samples of RNA or reverse transcriptase PCR and cDNA can be detected by methods described above. This procedure is also well known in the art.

[0094] An alternative means for determining the presence specific antibodies produced as a result of infection or exposure of the Hepatitis C virus is in situ hybridization, or more recently, in situ polymerase chain reaction. In situ PCR is described in Neuvo et al. [Neuvo, et al. (1993) American Journal of Surgical Pathology 17(7), 683-690.], Intracellular localization of polymerase chain reaction (PCR)-amplified Hepatitis C cDNA; Bagasra et al. [Bagasra, et al. (1992) J. New England Journal of Medicine 326(21):1385-1391.], Detection of Hepatitis C virus by in situ polymerase chain reaction; and Heniford et al. [Heniford, et al. (1993) Nucleic Acids Research 21(14):3159-3166.], Variation in cellular EGF receptor mRNA expression demonstrated by in situ reverse transcriptase polymerase chain reaction. In situ hybridization assays are well known and are generally described in Methods Enzymol. incorporated by reference herein. In an in situ hybridization, cells are fixed to a solid support, typically a glass slide. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of target-specific probes that are labeled. The probes are preferably labeled with radioisotopes or fluorescent reporters.

[0095] The above described probes are also useful for in-situ hybridization or in order to locate tissues which express this gene, or for other hybridization assays for the presence of this gene or its mRNA in various biological tissues. In-situ hybridization is a sensitive localization method which is not dependent on expression of antigens or native vs. denatured conditions.

[0096] Oligonucleotides are 3′end-labeled with [α-³⁵S]dATP to specific activities in the range of 1×10¹⁰ dpm/ug using terminal deoxynucleotidyl transferase.

[0097] Unincorporated labeled nucleotides are removed from the oligo probe by centrifugation through a Sephadex G-25 column or by elution from a Waters Sep Pak C-18 column.

[0098] A probe can be identified as capable of hybridizing specifically to its target nucleic acid by hybridizing the probe to a sample treated according the protocol of this invention where the sample contains both target virus and animal cells (e.g., nerve cells). A probe is specific if the probe's characteristic signal is associated with the Hepatitis C virus DNA in the sample and not generally with the DNA of the host cells and non-biological materials (e.g., substrate) in a sample.

[0099] The following stringent hybridization and washing conditions will be adequate to distinguish a specific probe (e.g., a fluorescently labeled DNA probe) from a probe that is not specific: incubation of the probe with the sample for 12 hours at 37° C. in a solution containing denatured probe, 50% formamide, 2× SSC, and 0.1% (w/v) dextran sulfate, followed by washing in 1× SSC at 70° C. for 5 20 minutes; 2× SSC at 37° C. for 5 minutes; 0.2× SSC at room temperature for 5 minutes, and H₂O at room temperature for 5 minutes. Those of skill will be aware that it will often be advantageous in nucleic acid hybridizations (i.e., In situ, Southern, or other) to include detergents (e.g., sodium dodecyl sulfate), chelating agents (e.g., EDTA) or other reagents (e.g., buffers, Denhardt's solution, dextran sulfate) in the hybridization or wash solutions.

[0100] It will be apparent to those of ordinary skill in the art that a convenient method for determining whether a probe is specific for a Hepatitis C viral nucleic acid against antibodies utilizes a Southern blot (or Dot blot) using DNA prepared from one or more Hepatitis C virus. Briefly, to identify a target specific probe DNA is isolated from the virus. Test DNA either viral or cellular is transferred to a solid (e.g., charged nylon) matrix. The probes are labeled following conventional methods. Following denaturation and/or prehybridization steps known in the art, the probe is hybridized to the immobilized DNAs under stringent conditions. Stringent hybridization conditions will depend on the probe used and can be estimated from the calculated T_(m) (melting temperature) of the hybridized probe (see, e.g., Sambrook for a description of calculation of the T_(m)).

[0101] For radioactively-labeled DNA or RNA probes an example of stringent hybridization conditions is hybridization in a solution containing denatured probe and 5× SSC at 65° C. for 8-24 hours followed by washes in 0.1×SSC, 0.1% SDS (sodium dodecyl sulfate) at 50-65° C. In general, the temperature and salt concentration are chosen so that the post hybridization wash occurs at a temperature that is about 5° C. below the T_(M) of the hybrid. Thus for a particular salt concentration the temperature may be selected that is 5° C. below the T_(M) or conversely, for a particular temperature, the salt concentration is chosen to provide a T_(M) for the hybrid that is 5° C. warmer than the wash temperature. Following stringent hybridization and washing, a probe that hybridizes to the Hepatitis C viral antibodies, as evidenced by the presence of a signal associated with the appropriate target and the absence of a signal from the non-target nucleic acids, is identified as specific for the Hepatitis C virus. It is further appreciated that in determining probe specificity and in utilizing the method of this invention to detect Hepatitis C viral specific antibodies, a certain amount of background signal is typical and can easily be distinguished by one of skill from a specific signal. Two fold signal over background is acceptable.

[0102] Screening Assays For Pharmaceutical Agents of Interest in Reducing Viral Load of Hepatitis C virus.

[0103] Hepatitis C virus drug screening assays which determine whether or not a drug may reduce the viral load described herein are contemplated in this invention. Such assays comprise incubating whole blood, lymphocytes, such as B cells, or PBMC with the mitogen and the Hepatitis C virus drug and evaluate whether the cell has antibodies produced against the Hepatitis C virus proteins or peptides and determining therefrom the effect of the compound on the activity of such agent. Alternatively, the anti-Hepatitis C agent is administered to the subject and either whole blood, lymphocytes, or PBMC is obtained and incubated with a mitogen so as to evaluate antibodies produced against Hepatitis C virus.

[0104] This invention is further illustrated in the Experimental Details section which follows. This section is set forth to aid in an understanding of the invention but is not intended to, and should not be construed to, limit in any way the invention as set forth in the claims which follow thereafter.

EXPERIMENTAL DETAILS EXAMPLE 1 Early Detection of Specific Antibodies to HCV

[0105] Exposure to Hepatitis C virus and the concomitant infection leads to the production of Hepatitis C virus specific antibodies. These Hepatitis C virus specific antibodies are an important tool for the detection both for diagnostic and for screening purposes of the viral infection/exposure.

[0106] Some viral exposures lead to a fast (1-2 weeks) seroconversion, yet others have a longer lag, or “window period” between exposure/infection and seroconversion. While different and complex immunological mechanisms, and virus-host relationships, determine the length of the window period, the immune system is usually introduced to the new viral antigens at a very early time post exposure/infection.

[0107] The purpose of this new method is to detect viral specific antibodies to which the immune system of a subject has already been exposed. This is achieved by driving, in vitro, the B cells in the blood sample to maturation and the production of the virus-specific antibodies even if the process is still has not started or is delayed in vivo.

[0108] Correct interpretation of antibody patterns in seropositive individuals is important in understanding the immunodominant epitope mapping. There are numerous immunologic epitopes encoded by the HCV genome, both in structural and nonstructural proteins. Infected individuals usually mount a broad humoral immune response to many antigens; however, no specific antibody pattern has been identified that can differentiate recovery from persistent HCV infection. In most prospectively followed patients with acute infection, antibodies to core (c22-3) and NS3 (c33-c) antigens develop before and at higher titers than antibodies to other antigens, although in some cases antibodies to NS4 (c-100-3) or to NS5 antigens appear first. No seroconversion pattern has been shown to predict outcome of infection. Immunodominant epitopes have been found within the N-terminus of the core antigen (aa 20 to 34) and within the NS4 region (aa 1712 to 1731), the latter corresponding recombinant antigens (C-22-3 and C-100-3, respectively). Most, if not all, chronically infected patients have antibodies against the hypervariable 5′ end of the E2/NS 1 gene product (gp70).

[0109] Furthermore, biopsy proven chronic hepatitis has been documented both in patients with seemingly acute self-limited hepatitis C and in anti-HCV positive blood donors with persistently normal ALT levels. The documented occurrence of transfusion-associated hepatitis (TAH) C virus in recipients of anti-HCV negative blood, the frequent loss of HCV-RNA from the serum of patients under interferon treatment with subsequent reappearance of viremia after interferon withdrawl, and the finding of HCV-RNA in the liver of patients with seronegative chronic hepatitis C suggest that neither normalization of ALT, disappearance of anti-HCV antibodies nor disappearance of serum HCV-RNA necessarily imply complete recovery. Therefore, it seems likely that persistent infection (whether active or quiescent) occurs in the majority of HCV-infected individuals.

[0110] Although the mechanism(s) underlying HCV persistence are unknown, some observations suggest potential strategies by which HCV might regulate its lytic potential and avoid detection and elimination by the host's immune system. Since HCV does not replicate through a DNA intermediate, its persistence cannot be explained in terms of latency through integration of viral genome into the host genome.

[0111] The currently available diagnostic armamentarium of HCV infection includes serologic assays based on recombinant protein and/or synthetic peptide-based antibody capture assays, gene amplification techniques for detection of HCV-RNA sequences in serum or liver, and immunohistochemical and “in situ” hybridization techniques for detection of HCV-antigens or HCV-RNA sequences in tissue. Tissue based techniques are in the early stages of development and available in only a limited number of research laboratories.

[0112] First generation anti-HCV assays detected antibodies to a single recombinant antigen (C-100-3), which corresponds to a small portion of the C′-terminus of NS3 and nearly all of the NS4 gene product expressed in yeast as a fusion protein.

[0113] The most extensively evaluated second generation tests (EIA-2) detect antibodies to the core recombinant protein C-22-3 and to a recombinant protein, C-200, representing a composite of recombinant proteins including C-33c (NS3) and C-100-3 (NS4).

[0114] EIA-2 increases seroconversion rates in acute, transfusion-associated, or sporadic NANB hepatitis by 10-20% and shortens the window period between disease onset and seroconversion by a mean of 8 weeks; in 70-80% of cases, EIA-2 can detect anti-HCV within 4 weeks of disease onset.

[0115] The most extensively evaluated “confirmatory assay” is a second generation recombinant immunoblot assay (RIBA-2, Chiron Corporation, Emeryville, Calif.).

[0116] This assay consists of a nitrocellulose strip tp which recombinant HCV proteins 5.1.1(NS4), C-100-3(NS4), C-33-c(NS3) and C-22-3(core), have been blotted as discrete bands, along two levels of human IgG and superoxide dismutase (SOD) control.

[0117] The most consistent IgM anti-HCV response is directed against the nucleocapsid (core) antigen, and in some instances is the first marker of active anti-HCV seroconversion. Although IgM reactivity is short-lived compared to IgG and hence IgM antibody can be utilized as an acute phase marker of HCV infection.

[0118] Sequence diversity in both structural and non-structural coding regions of different HCV isolates can result in false-negative PCR reactivity. Primers specific for the highly conserved 5′UTR should be used to avoid missing viremia due to sequence heterogeneity. The use of nested PCR (double OCR with nested primers), frequently used to increase sensitivity and to avoid the hybridization step, greatly increases the inherent risk of contamination and may yield amplificator products of the expected size that are nonspecific.

[0119] Method and Materials:

[0120] Heparinized blood samples from individuals that are followed due to a high risk of HCV infection were used in this experiment. One ml. of blood was mixed with 2 ml. of complete media, and the mitogen(PWM) was added. Tubes were cultured in 37° C. humidified C02 incubator. The culture fluid was tested for the presence of HCV specific antibodies using ELISA kit (Minoliga). The results are O.D. readings. The cut-off for this run was 0.392. The study center considers positive those that are at lease 1.2 of that cut-off (−0.470). The confirmation test was for structural and non structural antibodies (in serum only).

[0121] Results:

[0122] All samples in Table 1. are subjects from high risk groups for Hepatitis C virus. All antibodies were Hepatitis C virus specific. TABLE 1 MADA# Serology New-test Confirmation 5312 0.155 0.262 − 5313 0.449 0.500 − 5314 0.410 0.557 + 5315 0.159 0.165 un 5316 0.342 0.553 − 5317 0.502 1.015 − 5318 0.516 0.761 +/− 5319 0.246 0.498 +/− 5320 0.430 0.366 −

[0123] Sample 5319 is a clear example of better detection via culture than via serology. Since the cutoffwas close to 0.4 O.D. Sample 5314 is also a clear positive (also confirmed) only after culture.

EXAMPLE 2 Early Detection of Specific Antibodies of HCV Exposure

[0124] Further studies in population of individuals with an initial positive or boarder line ELISA in one test.

[0125] Results: TABLE 2 O.D. ratio in O.D. ratio in Donor No. serum supernatant Confirmation 5328 3.12 3.29 − 5329 1.93 1.88 + 5330 1.11 0.92 − 5331 5.51 2.14 + 5332 5.56 5.98 + 5321 1.70 1.99 − 5322 1.08 2.35 − 5323 0.58 0.30 un 5324 0.61 1.32 − 5325 1.10 2.01 − 5326 1.31 2.05 + 5327 3.62 5.90 +

[0126] Confirmation was only done using the structural and non structural protein antibody-test in the serum. Sample 5322, 5325, 5326 are examples of a positive diagnosis only after culture.

EXAMPLE 3 Multiple Antigen Assay

[0127] An antigen (on some type of a carrier) is inside the tube during the incubation period of the culture (blood culture).

[0128] The presence of the antigen serves mainly as a “detector” and “binder” (for ligand) for the antigen specific antibodies that are present (or being produced) in the culture. It could also serve at the same time as a specific stimulator (specific) of the specific segment of the immune process that is taking place in the tube.

[0129] The antigen may be in the tube prior to blood drawn in (or cells added) or added at any time-point during the culture. (Thus it could be in contact during the culture:days, hours or minutes). The antigen can be attached to (or bound to): 1) The tube itself either above the level of the culture liquid, at the level of the culture liquid, or below the level of the culture liquid; 2) bound to a nitrocellulose strip; 3)bound to any synthetic carrier (plastic of all sorts); 4) bound to beads, microspheres, etc; 5) be part of a “dry chemistry” (or more exact “dry immunochemistry” system [closed or open]). The surfaces of all the above, (especially #beads), can be smooth or grooved or in any other form of shape that will increase its surface area.

[0130] The antigen can be bound to all the above carriers directly or via carriers, “arms” and other methods that put a distance between the antigen and the “carrier's” surface. The antigen can be applied to the “carrier” at any shape or size: ring around the tube, a line, a dot, a shape of a + or + or P or any other letter or shape. The antigen can be attached to the carrier at one given concentration or at several concentrations, (either as discreet “sites” or as a continuous (or gradient). The antigen can be a few antigens—as more than one antigen can be applied to one tube (or carrier or bead or beads, etc.). The different antigens can be applied together (as a mixture) or in groups or individually. When applied to more than one spot, they can be applied using the same shape of mark or using a different one for each antigen.

[0131] The detection of the antibodies that bind to the antigen can be done by any of the “developing” and detection systems that are known to date (or that will be found later. The developing reagents can be in the tube initially or added at a later time. A very simple method will be to finish the development stage towards the end of the culture period or after the end of it all together. The development is either by direct binding of “tagged” antibodies or by competition assays. The “tag” can be an enzyme, a metal, a color colloid, (or fluorescent, illuminecent etc.) 

What is claimed is:
 1. A method for the detection of antibodies directed against Hepatitis C virus, comprising the following steps: a) obtaining a whole blood sample from the subject; b) incubating the whole blood sample in a culture in the presence of media containing mitogen, so as to induce polyclonal activation of lymphocytic cells to produce antibodies to the hepatitis c virus; c) exposing the resultant culture of step b) to a hepatitis c virus antigen, thereby allowing an antigen-antibody immune complex to form; and d) detecting the presence of antigen-antibody immune complex of step c); wherein the presence of hepatitis c virus specific antibodies is indicative of the subject being exposed to hepatitis C virus.
 2. The method of claim 1, wherein the culture of step c) results in a supernatant, and the supernatant is exposed to an antigen for the selected virus, thereby allowing an antigen-antibody immune complex to form.
 3. The method of claim 1, wherein the culture of step c) results in a cellular fraction which is exposed to an antigen, thereby allowing an antigen-antibody immune complex to form.
 4. The method of claim 1, wherein the mitogen activates lymphocytic cells.
 5. The method of claim 4, wherein the mitogen is pokeweed mitogen, a lectin, a bacterial endotoxin, a virus, lipid A, or a lymphokine.
 6. The method of claim 1, wherein the mitogen is pokeweed mitogen.
 7. A method of detecting a viral infection in a subject, comprising the following steps: a) obtaining a whole blood sample from the subject; b) incubating the whole blood sample in a culture in the presence of a media containing a mitogen, so as to induce polyclonal activation of lymphocytic cells to produce antibodies; c) treating the cells so as to separately recover nucleic acid sequences; d) contacting the nucleic acid sequences with single-stranded labeled oligonucleotide primers, the primers being capable of specifically hybridizing with a nucleic acid sequence for the Hepatitis C virus s antibody, under hybridizing conditions; e) amplifying any nucleic acid sequences to which a pair of primers hybridizes so as to obtain a double-stranded amplification product; and i) detecting the presence of the amplification product, the presence thereof being indicative of the presence of the Hepatitis C virus.
 8. The method of claim 7, wherein the nucleic acid sequence is DNA, RNA or cDNA.
 9. The method of claim 7, wherein the single-stranded oligonucleotide primers are labeled with biotin.
 10. The method of claim 7, wherein the single-stranded oligonucleotide probes are labeled with fluorescein.
 11. The method of claim 7, wherein the marker is alkaline-phosphatase.
 12. The method of claim 7, wherein the mitogen is an activator of lymphocytic cells.
 13. The method of claim 11, wherein the mitogen is pokeweed mitogen, a lectin, a bacterial endotoxin, a virus, lipid A, or a lymphokine.
 14. The method of claim 12, wherein the mitogen is pokeweed mitogen.
 15. A kit for the detection of Hepatitis C virus specific antibody from a subject, comprising: a container for collecting whole blood samples, wherein the container contains a media containing mitogen, effective to induce polyclonal activation of lymphocytic cells leading to antibody production and an assay for the detection of the Hepatitis C virus specific antibody.
 16. The kit of claim 15, wherein the assay is an enzyme linked immunosorbent assay, dot blot or an immunofluorescence assay.
 17. The kit of claim 16, wherein the container is made of a plastic, glass, or metal material.
 18. The kit of claim 18, wherein the container is a test tube or a flask.
 19. The kit of claim 18, wherein the container is vacuum sealed.
 20. The kit of claim 19, wherein an antigen is bound to the container.
 21. The kit of claim 20, wherein multiple antigens are bound to the container. 